OBJECTIVE We sought to use cardiac magnetic resonance (CMR) to establish sensitive and reliable indexes for noninvasive detection of acute cardiac allograft rejection. BACKGROUND Appropriate surveillance for acute allograft rejection is vitally important for graft survival. The current gold standard for diagnosing and staging rejection after organ transplantation is endomyocardial biopsy, which is not only invasive but also prone to sampling errors. The motivation of this study is to establish a CMR-based alternative that is noninvasive and sensitive for early detection of allograft rejection before irreversible damage occurs. METHODS We employed a noninvasive 2-pronged approach to detect acute cardiac allograft rejection using a rodent working heart and lung transplantation model. We used CMR to detect immune-cell infiltration at sites of rejection by monitoring the accumulation of dextran-coated ultra-small superparamagnetic-iron-oxide-labeled immune cells (in particular macrophages) in vivo. Simultaneously, we used CMR tagging and strain analysis to detect regional myocardial function loss resulting from acute rejection. RESULTS Immune cells infiltration, mainly macrophages and monocytes, could be identified with CMR by in vivo labeling with ultra-small superparamagnetic-iron-oxide. Our data show that immune-cell infiltration in cardiac allograft rejection was highly heterogeneous. Thus, it is not surprising to find inconsistencies between rejection and endomyocardial biopsy results because of the limited number and small samples available. Tagged CMR and strain analysis showed that, as with immune-cell infiltration, ventricular functional loss was also heterogeneous. Although changes in global systolic function were generally not observed until the later stages of rejection, our data revealed that a functional index derived from local strain analysis correlated well with rejection grades, which may be a more sensitive parameter for detecting early rejection. CONCLUSIONS CMR is noninvasive and provides a 3-dimensional, whole-heart perspective of the rejection status, potentially allowing more reliable detection of acute allograft rejection.